Our goal is to design and produce a clade C HIV MVA vaccine that will elicit antibodies capable of preventing HIV acquisition. Our clade B HIV VLP-expressing MVA-vectored vaccine has been tested clinically and shown good ability to Induce Env-specific antibodies. The clade B HIV vaccine, however, is not designed to express full length Env that we have found to prime the most protective antibody responses using the SIV rhesus macaque model (see Program Overview). Two technical approaches are proposed to increase the induction of protective Ab responses; the development of a MVA-vectored HIV vaccine encoding full-length, clade 0 gp160 and the use of MVA-expressed CD40L as a B-cell adjuvant. Specifically,we will 1) Construct a clade C VLP MVA vaccine expressing full-lengfh gp160 Env to optimize the protective avidity and neutralizing activity of elicited Env-specific Ab, 2) Insert target sequences for EB66 stem cellspecific miRNAs into 3' untranslated regions of vaccine Env inserts to suppress insert expression and enhance titers and vector stability during manufacture in the EB66 cell line and 3) Construct CD40Lexpressing MVA to serve as a strong B cell adjuvant for the titer and avidity of Env-specific Ab. Production of MVA vaccines is a challenge because of the growth of these vaccines in chick embryo fibroblasts that require sourcing and producing large batches of primary cells. Because of this, GeoVax has explored the use of continuous cell lines for growth of MVA and has found the EB66 duck stem cell-derived line developed by Vivalis LLC. Nantes France to be a promising option. GeoVax has produced dedicated Master and Working EB66 cell banks. The goals for this project include 1) Qualification of media and production processes for EB66 cells as the MVA manufacturing cell substrate focusing on end-of-production testing and the generation of Master seed stocks, 2) Production of a GMP-like pilot/feasibility lot of MVA-vectored clade C vaccine and the MVA encoding CD40L to validate the manufacturing process and to produce material for animal safety testing and 3) Production of a GMP lot of each vaccine to be used in Phase 1 clinical testing by the HVTN.